Prolonged in vivo imaging of Xenopus laevis

Abstract

Background: While live imaging of embryonic development over long periods of time is a well established method for embryos of the frog Xenopus laevis, once development has progressed to the swimming stages, continuous live imaging becomes more challenging because the tadpoles must be immobilized. Current imaging techniques for these advanced stages generally require bringing the tadpoles in and out of anesthesia for short imaging sessions at selected time points, severely limiting the resolution of the data. Results: Here we demonstrate that creating a constant flow of diluted tricaine methanesulfonate (MS-222) over a tadpole greatly improves their survival under anesthesia. Based on this result, we describe a new method for imaging stage 48 to 65 X. laevis, by circulating the anesthetic using a peristaltic pump. This supports the animal during continuous live imaging sessions for at least 48 hr. The addition of a stable optical window allows for high quality imaging through the anesthetic solution. Conclusions: This automated imaging system provides for the first time a method for continuous observations of developmental and regenerative processes in advanced stages of Xenopus over 2 days.

3D-printable tools for developmental biology: Improving embryo injection and screening techniques through 3D-printing technology

“…These molds facilitated the screening and imaging process, making it easier and faster to generate high quality (and aesthetically pleasing) data while critically allowing for the recovery of the Xenopus tadpoles from the anesthesia in all the cases. We designed a screening stamp to fit Xenopus tadpoles at stage 43, keeping in mind that they do not survive long-term exposure to anesthesia when in a fixed positioned [7]. Correctly orienting and immobilizing the embryos for phenotypic comparison becomes a countdown task, but using our screening tool makes differences between tadpoles readily apparent, crucial for fast and accurate screening under severe time pressure.…”

“…Swimming tadpoles need to be anesthetized with MS-222 [7,10]. The two by two-sided mold for Xenopus is generated by firmly pressing against the flattened modeling clay (wax based clay) in a p35 petri dish.…”

“…We anesthetized the animals directly on the dish, where they were oriented and imaged. Then we recovered the tadpoles on a rocker in a new dish [7]. For a more high-throughput screening process, a multimold Xenopus tropicalis tadpole stamp was designed, to simultaneously screen 20 (4 x 5) tadpoles, in a drastically more efficient process.…”

“…Depending on the lab, swimming tadpoles need to be anesthetized with MS-222, which after 10-15 minutes kills the animal, and screened by eye in a dish, and imaged in a hand-sculpted well in a clay dish [7]. The stamp (Fig.…”

“…These studies generally require the injection of reagents such as dextran, diI, morpholinos, RNAi, mRNA in vitro synthesized or constructs inside vectors [3,4,5,6]. Subsequently, checking the embryos, or the frequently highly mobile juveniles, is an essential next step for correctly describing a phenotype or a whole in vivo process [7,8]. Generally, each individual laboratory has its own special injection and screening system.…”